Christina Tröls DIVERSITY OF MICROBIAL ENDOPHYTES FROM WARBURGIA UGANDENSIS Masterarbeit Zur Erlangung des akademischen Grades einer Magistra an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz Priv.-Doz. DI Dr. Angela Sessitsch AIT Austrian Institute of Technology A-2444 Seibersdorf 2009 Danksagung Diese Masterarbeit ist am AIT Austrian Institue of Technology in Seibersdorf entstanden. Besonders möchte ich mich für die freundliche Arbeitsatmosphäre in der Abteilung für Bioresources, vor allem im „Dissertantenzimmer“, bedanken. Claudia Fenzl, Melanie Kuffner, Patrick Domnanich, Valerie Hubalek, Kuheli Das Gupta, Tanja Garcia-Liberos und nicht zuletzt Marlies Czetina standen mir immer mit Rat und Tat zur Seite. Ich werde euch sehr vermissen! Danke für eure Unterstützung und Motivation während dieser Arbeit und nicht zu vergessen für unsere interessanten Gespräche in den Kaffeepausen. Außerdem möchte ich mich bei meinen Betreuerinnen Priv.-Doz. DI Dr. Angela Sessitsch und Mag. Dr. Birgit Mitter bedanken, die mir engagiert zur Seite standen und ohne die diese Arbeit nicht möglich gewesen wäre. Besonderer Dank gilt meinen Eltern, Annegret und Werner Tröls und meinen drei Schwestern und ihren Familien, denen ich sehr verbunden bin. Vor allem ist es mir auch ein Anliegen, mich für die liebevolle Unterstützung von meinem Freund Kurt Matoy, meiner Zwillingsschwester Martina Tröls und meinen guten Freundinnen Andrea Schuster und Ursula Lemmerer zu bedanken. 1 CONTENTS 1 AIM OF THE THESIS ................................................................................3 2 INTRODUCTION........................................................................................4 2.1 WARBURGIA UGANDENSIS – THE AFRICAN PEPPER BARK TREE ............................................................ 4 2.2 DRIMANE SESQUITERPENES.................................................................................................................. 5 2.3 ENDOPHYTES ........................................................................................................................................ 5 2.4 CULTIVATION-INDEPENDENT MICROBIAL COMMUNITY ANALYSIS ........................................................ 7 2.4.1 RFLP analysis ................................................................................................................................. 8 2.4.2 Terminal restriction fragment length polymorphism analysis......................................................... 8 2.4.3 rDNA cloning and sequencing ........................................................................................................ 8 3 MATERIAL AND METHODS.................................................................10 3.1 LIST OF MATERIALS............................................................................................................................ 10 3.1.1 Instruments and Devices ............................................................................................................... 10 3.1.2 Media ............................................................................................................................................ 10 3.1.3 Restriction enzymes....................................................................................................................... 11 3.1.4 Kits and Protocols......................................................................................................................... 11 3.2 PLANT MATERIAL AND SAMPLE SITE ................................................................................................... 11 3.3 DNA ISOLATION ................................................................................................................................. 13 3.4 DETERMINATION OF THE PRESENCE OF CHLOROPLAST DNA IN THE DNA SAMPLES........................... 13 3.5 T-RFLP COMMUNITY FINGERPRINTING .............................................................................................. 14 3.5.1 PCR ............................................................................................................................................... 14 3.5.2 PCR product purification.............................................................................................................. 17 3.5.3 T-RFLP.......................................................................................................................................... 17 3.5.4 rDNA clone library construction................................................................................................... 18 3.5.5 RFLP analysis ............................................................................................................................... 21 3.5.6 Sequence analysis.......................................................................................................................... 21 4 RESULTS....................................................................................................23 4.1 DETERMINATION OF THE PRESENCE OF CHLOROPLAST DNA IN THE DNA SAMPLES........................... 23 4.2 T-RFLP ANALYSIS.............................................................................................................................. 24 4.2.1 Bacterial community structure ...................................................................................................... 24 4.2.2 Fungal community structure ......................................................................................................... 26 4.2.3 Structural diversity........................................................................................................................ 36 4.3 SEQUENCE ANALYSIS IN WARBURGIA UGANDENSIS ........................................................................... 41 4.3.1 Sequence analysis of uncultured bacterial endophytes in Warburgia ugandensis..................... 41 4.3.2 Sequence identification of uncultured bacterial endophytes in Warburgia ugandensis................ 45 4.3.3 Sequence analysis of uncultured fungal endophytes in Warburgia ugandensis............................ 46 4.3.4 Sequence identification of uncultured fungal endophytes in Warburgia ugandensis.................... 50 5 DISCUSSION AND CONCLUSIONS .....................................................52 6 REFERENCES ...........................................................................................55 2 1 Aim of the thesis This diploma thesis is part of an ongoing research project (FWF P19851-B17) that addresses endophytic communities of the tropical tree Warburgia ugandensis which is highly regarded as remedy of various human diseases but mainly for Malaria caused by bacteria, fungi or viruses in traditional folk medicine. Phytochemical studies on W. ugandensis plants led to the identification of drimane sesquiterpenes and flavonol glycosides (Manguro et al., 2003) with broad biological activities. It can be expected that, simultaneously with this high plant physiological activity, associated endophytic bacteria also proliferate and exhibit broad functional diversity. Under these hostile conditions bacterial populations colonizing W. ugandensis should predominantly consist of species that evolved genetic mechanisms to overcome the toxic effects of the plant secondary metabolites. Consequently, the research- project aims at investigating the composition of W. ugandensis metabolites, the microbial community composition and at analyzing the genetic adaptation of the endophytes to their environment. The aim of the thesis was to elucidate the community structure of uncultured microbial endophytes (bacteria and fungi) in W. ugandensis trees and to identify the dominant microbial groups. To achieve this goal cultivation-independent community analysis was performed employing terminal restriction length polymorphism (T-RFLP) analysis as well as ribosomal DNA-PCR cloning and sequencing. 3 2 Introduction 2.1 Warburgia ugandensis – the African pepper bark tree Warburgia ugandensis Sprague [=W. salutaris (Bertol.f.) Chiov], also called pepper bark tree is endemic in evergreen forests and woodland ravines of northern KwaZulu-Natal, Swaziland, Mpumalanga, Uganda, and Kenya. The genus of the pepper bark tree, which belongs to a small family of tropical trees named Canellaceae, is represented by two species W. ugandensis Sprague (Figure 1) and W. stuhlmannii Engl.. These species are distinguishable in their size of their leaves and in the colour of their stem bark (Dale and Greenway, 1961; Beentje, 1994). Figure 1: Pictures of W. ugandensis tree depicting a young tree (left) an extensively harvested stem for medicinal purposes (middle) and the leaves with emerging flower buds (right). In wide areas of eastern Africa, where W. ugandensis grows (Beentje, 1994; Dale and Greenway, 1961), people use the powdered bark for the treatment of a broad range of human diseases (stomach-ache, constipation, toothache, common cold, cough, fever, muscle pains, weak points, measles and malaria (Beentje, 1994; Kokwaro, 1976)). The application is done orally (aqueous infusion, smoke) or as an ointment by mixing the powder with fat. The healing effects originate from the high content of drimane sesquiterpenes. The most comprehensive phytochemical investigations of drimane sesquiterpenes of the stem bark of W. ugandensis were carried out by Kioy et al. (1990) and Wube et al. (2005). 4 2.2 Drimane Sesquiterpenes Drimane sesquiterpenes belong to the class of terpenoids and 150 different compounds are known up to